This invention relates to the use of aggregated protein-polycationic polymer conjugates in genetic vaccines. More particularly it relates to the use of macroaggregated albumin conjugates for oral delivery of vaccines.
The mucosal immune system is extremely important in human resistance to microbial pathogens, but complicating the picture is the role of the mucosal immune system in suppressing undesirable antigenic responses to inhaled and ingested antigens in order to avoid massive allergic sensitivity (Mowat, 1987). In addition, systemic immunization usually does not elicit significant mucosal immune responses (Mestecky et al., 1994). In the context of HIV, the genital surface is the most important target tissue for immunity, and it is particularly difficult to immunize directly, especially in the male. Hence, an HIV vaccine aimed at inducing mucosal immunity will almost certainly have to be administered elsewhere (e.g., the gut), but it must still result in the accumulation of immune effector cells and molecules in the genital mucosa. Airway immunization (e.g., intranasal and intratracheal) and gut immunization have both been shown to produce responses with effector cells spilling over to other mucosal surfaces, especially the genital tract. Recent work has revealed that antigens (and particles) are delivered across the mucosal surface relatively unchanged to antigen presenting cells underneath the surface via specialized cells (membranous cells or M cells) that are derived from the normal epithelium. It is generally believed that antigens taken up by the M cells, which cover the mucosal inductive sites, are channeled to parenchymal macrophages, dendritic cells, B lymphocytes and even mast cells. Under some conditions, such as viral infection, or perhaps genetic immunization, the antigen can be processed and perhaps presented directly by the epithelial cells to the underlying B and T cells. Hence, regardless of how the antigen is administered, antigen (or antigen expressed via plasmid DNA) that can be delivered to these antigen presenting cells should result in mucosal immune responses.
In genetic immunization, a simple mammalian expression plasmid containing the gene for an antigen is administered to the animal rather than the antigen itself. Expression of the antigen gene in this manner produces the antigen intracellularly, making it a substrate for major histocompatibility (MHC) class I presentation (Schirmbeck et al., 1995) thereby enabling strong cellular immune responses to be produced. The mechanisms of antigen presentation include transfection and expression of the antigen within professional antigen presenting cells (e.g., macrophages or dendritic cells), or transfer of peptides from a different expressing cell (e.g., cytoplasm transfer from muscle cells) to a professional antigen presenting cell (Doe et al., 1996, Huang et al., 1994). Anti-HIV envelope cytotoxic T-lymphocytes (CTL) have been induced in mice and nonhuman primates by genetic immunization (Wang et al., 1994). Furthermore, intramuscular genetic vaccination with influenza nucleoprotein expressing plasmids has produced long-lived CD8+ CTL mediating cross-strain protection against flu (Ulmer et al., 1994). Humoral responses, e.g., to HIV-1 envelope protein (Wang et al., 1993, Lu et al., 1995), are also produced by such immunizations, either by the target cells themselves acting as antigen presenting cells, or by shedding of the protein products from the cell surface and subsequent processing of the peptides by professional antigen presenting cells through the MHC class II pathway. In fact, repeated intramuscular injections of plasmids have resulted in a conversion of the dominant CTL response into a humoral response, with waning of the cellular cytotoxicity response in some reports (Fuller et al., 1994). Hence, the appropriate dose, timing, and route(s) for administration are critical to producing an optimal genetic immunization response.
Inefficient DNA delivery remains one of the main impediments to successful gene therapy and DNA immunizations (Thierry et al., 1997). Transfection in vivo has been accomplished using cationic lipid/DNA complexes administered by injection intravenously (Zhu et al., 1993) and intramuscularly (Mitchell et al., 1995), and by application to mucosal surfaces (Schmid et al., 1994). Naked DNA has also been used intravenously, but it has a very brief half-life (Lew et al., 1995 and Kawabata, et al. 1995) and thus is extremely inefficient. However, intramuscular and intradermal injection of naked DNA has been effective in a variety of animal models. Viral vectors can be very efficient transiently, but the induction of antiviral immune responses prevents repeated administrations and may result in lowered transgene expression (Weichselbaum et al., 1997). In addition, toxicity may result from either cationic lipids or viral vectors in some cases. Microencapsulated plasmids containing reporter genes given orally penetrate the gastrointestinal tract surfaces and cause expression of the foreign genes in the cells of the gut and associated lymphoid tissue (Mathiowitz et al., 1997). Microspheres can also penetrate lymphoid tissue given by other routes as well, as has been observed in studies of protein antigen delivery (Marx et al., 1993 and Jenkins et al., 1995). Delivery of DNA to cutaneous tissues using a gene gun (Williams et al., 1991) has produced humoral systemic immunization (Tang et al., 1992) and cellular responses that were protective in the lung against Mycoplasma pulmonis infection (Barry et al., 1995). Despite all of these different, methods, none has proved to be broadly efficient in gene transfection application in vivo.
Mucosal immunity has been induced by nasal administration of plasmid DNA expression vectors, e.g., protective immunity to flu virus challenge with a vector encoding the influenza hemagglutinin protein (Fynan et al., 1993). Packaging with viral vectors (e.g., adeno-associated viruses), or incorporating antigenic genes into vaccinia virus has been frequently used, but has the disadvantage of inducing an immune reaction to the associated viral proteins as well, limiting the potential for boosting, or for administration of other antigens by the same route.
Expression library immunization (ELI) is a novel vaccine approach that weds the power of recombinant DNA technology to genetic immunization (Barry et al., 1995). In ELI, a pathogen""s genome is broken into small fragments and cloned into mammalian expression plasmids to create a vaccine representing all or most of the antigens of the pathogen. The expression plasmids contain a strong promoter (e.g., CMV) and expression of the protein products can be controlled by fusion to targeting sequences, i.e., the carboxy terminus of ubiquitin, which directs the protein to the proteasome for MHC class I presentation, a signal sequence for secretion, which directs the product to be secreted for antibody induction for MHC class II presentation, or without an additional sequence, which the protein is processed based on the native properties of the protein sequence. ELI was first demonstrated effective against the bacterial pathogen Mycoplasma pulmonis by Michael Barry and colleagues (Barry et al., 1995). Expression libraries were generated by fusing fragments of M. pulmonis genomic DNA to the carboxy-termini of human growth hormone (containing a signal sequence for secretion) or ubiquitin. Ubiquitin fusions were recently reported to produce CTL, but reduce antibody induction (Gillanders et al., 1997); however, gene gun vaccination with these ELI vaccines for M. pulmonis produced both CTL and antibody protection against mycoplasma infection at least an order of magnitude better than the best traditional mycoplasma vaccine available. ELI appears useful for generating multiple immune responses simultaneously, in a manner analogous to that of a live/attenuated pathogen vaccine. Given the pattern of responses seen in the resistant sexually exposed individuals in HIV infections, the broad range of antigen specificities that can be delivered by the expression library approach is particularly attractive. ELI vaccines are also safer than live-attenuated vaccines, because the genome of the pathogen is fragmented and scattered across large numbers of separate plasmids precluding reassembly of a pathogenic viral genome.
This invention demonstrates for the first time the use of an oral delivery system for a genetic vaccine in which the DNA is conjugated to an aggregated protein and a polycationic polymer. It is noteworthy that although the prior art in genetic vaccines have utilized other modes of delivery, the strategy of using oral delivery for a DNA vaccine has not been developed, suggesting that this invention is indeed nonobvious.
An embodiment of the present invention is a composition comprising an expression vector bound to an aggregated protein-polycationic polymer conjugate, wherein the expression vector comprises a promoter polynucleotide sequence operatively linked to a polynucleotide sequence encoding an antigen.
In a specific embodiment of the present invention, the polynucleotide sequence encoding the antigen is a fragment of a genome or gene selected from the group of genomes or genes associated with a disease consisting of infectious disease, cancer and autoimmune disease.
In another specific embodiment of the present invention, the polynucleotide sequence encoding the antigen is a fragment of a genome or gene selected from the group of pathogenic genomes consisting of virus, bacterium, fungus and protozoa. In specific embodiments, the polynucleotide sequence encoding the antigen is a fragment of a genome selected from the group viral genomes consisting of human immunodeficiency virus (HIV), herpes simplex virus (HSV) hepatitis C virus (HCV), influenza virus and respiratory syncytial virus (RSV).
In a specific embodiment of the present invention, the polynucleotide sequence encoding the antigen is a fragment of a gene selected from the group of genes associated with an autoimmune disease consisting of rheumatoid arthritis, vaculitis, and multiple sclerosis.
A further embodiment of the present invention is a composition wherein the expression vector comprises a heterologous mammalian targeting sequence. In specific embodiments, the mammalian targeting sequence is ubiquitin or a signal sequence for secretion.
Another specific embodiment of the present invention is a method of producing a DNA vaccine comprising the step of incubating an expression vector with an aggregated protein-polycationic polymer conjugate to form DNA particles wherein the expression vector comprises a promoter polynucleotide sequence operatively linked to a polynucleotide sequence encoding an antigen. In specific embodiments, the DNA vaccine is administered to a mucosal (e.g., intranasal surface, oral surface, gastrointestinal surface and genitourinary tract surface) or parenteral surface (e.g., intraperitoneal, intravenous, subcutaneous, intramusclar and intradermal) of an organism. Organisms that may be treated using the method of the invention include, but are not limited to humans, cows, horses, pigs, dogs, cats, sheep goats, rabbits, rats, mice, birds, monkeys, chickens or fish.
An additional embodiment of the present invention is the method of inducing an immune response in an organism comprising the step of administering to an organism the expression vector bound to an aggregated protein-polycationic polymer conjugate wherein the expression vector is comprises a promoter polynucleotide sequence operatively linked to a polynucleotide sequence encoding an antigen.
In specific embodiments, the method of inducing an immune response comprises the step of co-administering to an organism the expression vector bound to an aggregated protein-polycationic polymer conjugate and a cytokine expression vector.
In further embodiments, the method of inducing an immune response comprises the step of administering to an organism one expression vector bound to an aggregated protein-polycationic polymer conjugate, wherein the expression vector comprises a promoter polynucleotide sequence operatively linked to a first polynucleotide sequence encoding an antigen and a second polynucleotide sequence encoding a cytokine. In specific embodiments, the first and second polynucleotide sequences are under transcriptional control of the same promoter polynucleotide sequence. In other embodiments, the first and second polynucleotide sequences are under transcriptional control of different promoter polynucleotide sequences. One skilled in the art realizes that the polynucleotide sequences may be in tandem under control of the same promoter sequence or the polynucleotides are under control of separate promoter sequences.
A further embodiment of the present invention is the method of introducing genes into a cell comprising the steps of forming a DNA particle comprising an expression vector bound to an aggregated protein-polycationic polymer conjugate wherein the expression vector comprises a promoter polynucleotide sequence operatively linked to a polynucleotide sequence encoding an antigen, and incubating the cells with the DNA particle under conditions wherein the cells take in the DNA particle.
Another embodiment of the present invention is a composition comprising an expression vector incubated with a protein-polycationic polymer suspension, wherein the expression vector comprises a promoter polynucleotide sequence operatively linked to a polynucleotide sequence encoding an antigen.
Yet further, an additional embodiment of the present invention is the method of inducing an immune response in an organism comprising the step of administering to an organism an expression vector incubated with a protein-polycationic polymer suspension, wherein the expression vector is comprises a promoter polynucleotide sequence operatively linked to a polynucleotide sequences encoding an antigen.
Other and further objects, features and advantages would be apparent and eventually more readily understood by reading the following specification and by reference to the company drawings forming a part thereof, or any examples of the present preferred embodiments of the invention are give for the purpose of the disclosure.